Fluid transport in insects is dependent on hydrogen ions. Vacuolar-type H+-ATPase pumps energize insect epithelial membranes in a manner similar to that of the ubiquitous Na/K-ATPase pump. Insects secrete fluid from hemolymph through cells into the lumen of the Malpighian tubule. The energy derived from the H + -ATPase pump allows for secondary transport that leads to this transport of fluid. The secretion of fluid is the first step in generation of urine in insects and regulation of the composition of their extracellular fluid. Despite the crucial role played by the H+-ATPase in urine formation, no information to date is available as to the nature of intracellular regulation of hydrogen ions in Malpighian tubules. Questions which arise are: What is the source of the hydrogen ions for the H+-ATPase? How is the pH of the Malpighian tubule cell cytoplasm maintained? Which acid/base transporter is activated during diuresis? The goals of the proposed research are to 1) determine the primary mechanism(s) of intracellular hydrogen ion homeostasis m isolated Mosquito Malpighian tubules and 2) determine the influence of intracellular and extracellular messenger molecules including cyclic AMP and diuretic hormones on intracellular hydrogen ion homeostasis. Preliminary data indicate that mosquito Malpighian tubules regulate their intracellular pH through at least two acid extrusion mechanisms, and two acid loading mechanism. Additionally, intracellular hydrogen and bicarbonate generation are under the control of carbonic anhydrase. Finally, cyclic AMP increases acid extrusion after an acute acid load suggesting a link between acid extrusion and cAMP-mediated diuresis. The proposed experiments will measure fluid secretion and hydrogen ion secretion rates in isolated mosquito Malpighian tubules in order to establish dose response relationships between inhibition of specific acid-base transporters and fluid and hydrogen ion secretion rates in insects. Intracellular pH will be measured using micro-fluorometric techniques which allow for real time recording of changes in quantities of cellular hydrogen ions. We will characterize the effects of inhibition and stimulation of the acid extruding and acid loading mechanism with respect to steady-state intracellular pH, recovery rates after acute acid loading, acid or base flux rates and Km and Vmax of the transporters. The results from the proposed experiments will, for the first time, describe in detail the underlying mechanism(s) for fluid secretion in Malpighian tubules. This body of knowledge will lead to potential insight into physiological processes that enable mosquitoes to be effective in resisting pesticide intervention and efficient vectors of pathogens.